Background Aspirin-exacerbated respiratory disease (AERD) differs from aspirin tolerant disease due in part to eosinophilic tissue infiltration and over-expression of arachidonic acid metabolic pathway components that lead to enhanced secretion of cysteinyl leukotrienes (CysLT) and prostaglandin D2 (PGD2) observed constitutively and, paradoxically, in response to aspirin and other cyclooxygenase inhibitors. We have previously demonstrated the capacity of interferon (IFN)-γ to drive CysLT expression and response. Objective We investigated eosinophils as a source for PGD2 production in AERD. Methods Eosinophils were enriched from tissue and peripheral blood obtained from control, aspirin tolerant, and AERD subjects. mRNA was extracted and evaluated for expression of hematopoietic PGD synthase (hPGDS). Expression of hPGDS protein was confirmed with western hybridization and immunofluorescence staining. Cells were stimulated with aspirin and secretion of PGD2 quantified. CD34+ progenitor cells were isolated and matured into eosinophils in the presence or absence of IFN-γ and hPGDS mRNA and PGD2 release measured. Results Gene expression analysis revealed that eosinophils from AERD tissue and blood display increased levels of hPGDS compared with asthmatic and control samples. Western hybridization confirmed the increase in hPGDS mRNA translated to increased protein expression. Immunofluorescence confirmed mast cells and eosinophils from AERD and asthmatic tissue demonstrated hPGDS expression with higher levels in AERD eosinophils. Incubation of eosinophils from blood and tissue with aspirin stimulated PGD2 release. IFN-γ-matured eosinophil progenitors showed enhanced hPGDS expression and increased levels of PGD2 release at baseline and following stimulation with aspirin. Conclusions In addition to mast cells, eosinophils represent an important source of PGD2 in AERD and identify a new target for therapeutic intervention. Clinical Implications Demonstration of eosinophils as a source of PGD2, that has been shown to be involved in the presence and severity of AERD, identifies a new target for therapeutic intervention.
Thymidine kinase-1 (TK1) is an important cancer biomarker whose serum levels are elevated in early cancer development. We developed a microchip electrophoresis immunoaffinity assay to measure recombinant purified TK1 (pTK1) using an antibody that binds to human TK1. We fabricated poly(methyl methacrylate) microfluidic devices to test the feasibility of detecting antibody (Ab)-pTK1 immune complexes as a step towards TK1 analysis in clinical serum samples. We were able to separate immune complexes from unbound antibodies using 0.5X phosphate buffer saline (pH 7.4) containing 0.01% Tween-20, with 1% w/v methylcellulose that acts as a dynamic surface coating and sieving matrix. Separation of the antibody and Ab-pTK1 complex was observed within a 5 mm effective separation length. This method of detecting pTK1 is easy to perform, requires only a 10 μL sample volume, and takes just 1 minute for separation.
Cancer is the second biggest killer in the United States. It has long been recognized that people differ in their susceptibility to many different types of cancer. Some of these differences are the heritable traits that modify the effects of environmental exposures. Genetic instability, which drives tumorigenesis, is itself fuelled by DNA damage and often by errors made by the DNA repair machinery. DNA repair, using natural sources of antioxidants, is a new developing area of cancer prevention. The aim of the current study was to investigate DNA repair capacity and DNA damage of Burkitt's lymphoma (Raji) cells exposed to Resveratrol and different extracts of Nagami kumquat (Fortunella margarita). Nagami kumquat is known as “the little gold gem of the citrus family,” and its pulp has been recently highlighted as one containing the most potent antioxidant and high phenolic contents among citrus fruits. Kumquat skin had the highest vitamin C content (3,069.183 μg/g) when compared to both the seed (506.14 μg/g) and pulp (2,440.448 μg/g). Recent evidence has shown that diets rich in antioxidants, such as those including kumquats, may be effective in lowering an individual's risk for cancer. We measured the DNA repair rate of cells by the Comet Assay, taken at several time points throughout the course of 1 hour. DNA damage was induced by exposing the cells to 4mM H2O2 for 15 minutes. This damage was then measured by the comet assay. We measured the amount of DNA damage at 0, 6, 15, 30 and 60 minutes, in order to determine the DNA repair rate. The results were analyzed with Tri-Tek Comet Score Software Version 1.5. The DNA Repair after one hour, using the kumquat Pulp was highly significant (P<0.05) according to the percentages of the DNA in the tail (3.72%) followed by skin (3.37%) and seed (2.28%). The results indicate that among citrus fruits, Kumquat as an antioxidant has a significant capacity to repair DNA. We were able to observe the effect of the Resveratrol (RV) on the rate of DNA repair which showed a highly significant difference when RV was incubated with the cells for a period of 24 hours compared to the non-treated controls (p<0.001). The DNA repair rate was shorter in RV at 15 minute exposure showing 1.09% compared to the non-treated group at 30 minutes which showed 1.175%. We also compared RV to the kumquat extracts and we found that there was a significant difference between the rates of DNA repair, showing that RV was significantly better compared to kumquat extracts. We can conclude that RV has three times higher DNA repair activity than extracts of Nagami kumquat. Citation Format: Albert V. Helsing, Atif El-Naggar, Philip M. Lynch, Michael Xiao, Madison Ramsden, B Fabrizio Alegre, Richard A. Robison, Kim L. O'Neill. The potential of Resveratrol and Nagami kumquat extracts in facilitating DNA repair in Raji cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1288. doi:10.1158/1538-7445.AM2013-1288
Lung cancer is the number one cause of cancer mortality and early detection will increase the survival of this disease. Thymidine kinase 1 (TK1) is a biomarker indicative of cell proliferation that is elevated in numerous malignancies including lung cancer. TK1 has been found to be elevated early in the development of lung cancer and therefore is an ideal target as an early detection biomarker. We have developed an enzyme-linked immunosorbent assay (ELISA) to detect TK1 in serum. Forty patients with pulmonary nodules and 18 healthy control individuals had serum collected under standard conditions, prior to operative removal of any lesions. All serum samples were analyzed using TK1 radioassay to measure TK1 activity, and TK1 ELISA to measure TK1 concentration. TK1 was significantly elevated in all patients with confirmed lung cancer. This was true even in stage IA, where patients (n=16) had significantly higher TK1 levels than controls. The clearest cut-off was 4.9 nM with an area under the curve of 0.792 for early stage lung cancer. The corresponding sensitivity and specificity was 82.4 and 83.3, respectively. TK1 concentration was a more sensitive and accurate indicator of lung cancer than TK1 activity. Overall, TK1 is significantly elevated in serum of lung cancer patients. This TK1 ELISA is sensitive and specific for detection of lung cancer and further trials with a larger patient base can confirm the potential of this method of detection. Citation Format: Madison K. Ramsden, Melissa M. Alegre, Michael J. Weyant, Daine T. Bennett, Jessica A. Yu, Atif Elnaggar, Richard A. Robison, Kim L. O'Neill. Detection of early-stage lung cancer using a novel ELISA for thymidine kinase 1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1866. doi:10.1158/1538-7445.AM2014-1866
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